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Title: Two phenylalanines in the C-terminus of Epstein-Barr virus Rta protein reciprocally modulate its DNA binding and transactivation function

Abstract

The Rta (R transactivator) protein plays an essential role in the Epstein-Barr viral (EBV) lytic cascade. Rta activates viral gene expression by several mechanisms including direct and indirect binding to target viral promoters, synergy with EBV ZEBRA protein, and stimulation of cellular signaling pathways. We previously found that Rta proteins with C-terminal truncations of 30 aa were markedly enhanced in their capacity to bind DNA (Chen, L.W., Chang, P.J., Delecluse, H.J., and Miller, G., (2005). Marked variation in response of consensus binding elements for the Rta protein of Epstein-Barr virus. J. Virol. 79(15), 9635-9650.). Here we show that two phenylalanines (F600 and F605) in the C-terminus of Rta play a crucial role in mediating this DNA binding inhibitory function. Amino acids 555 to 605 of Rta constitute a functional DNA binding inhibitory sequence (DBIS) that markedly decreased DNA binding when transferred to a minimal DNA binding domain of Rta (aa 1-350). Alanine substitution mutants, F600A/F605A, abolished activity of the DBIS. F600 and F605 are located in the transcriptional activation domain of Rta. Alanine substitutions, F600A/F605A, decreased transcriptional activation by Rta protein, whereas aromatic substitutions, such as F600Y/F605Y or F600W/F605W, partially restored transcriptional activation. Full-length Rta protein with F600A/F605A mutations weremore » enhanced in DNA binding compared to wild-type, whereas Rta proteins with F600Y/F605Y or F600W/F605W substitutions were, like wild-type Rta, relatively poor DNA binders. GAL4 (1-147)/Rta (416-605) fusion proteins with F600A/F605A mutations were diminished in transcriptional activation, relative to GAL4/Rta chimeras without such mutations. The results suggest that, in the context of a larger DBIS, F600 and F605 play a role in the reciprocal regulation of DNA binding and transcriptional activation by Rta. Regulation of DNA binding by Rta is likely to be important in controlling its different modes of action.« less

Authors:
 [1]; ;  [2]; ;  [3];  [4];  [2]
  1. Department of Respiratory Care, Chang Gung Institute of Technology, Chaiyi, Taiwan (China)
  2. Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, CT 06520 (United States)
  3. Department of Pediatrics, Yale University School of Medicine, Room 420 LSOG, 333 Cedar St., New Haven, CT 06520 (United States)
  4. German Cancer Research Center, Department of Tumour Virology, Im Neuenheimer Feld 242, Heidelberg (Germany)
Publication Date:
OSTI Identifier:
21357498
Resource Type:
Journal Article
Journal Name:
Virology
Additional Journal Information:
Journal Volume: 386; Journal Issue: 2; Other Information: DOI: 10.1016/j.virol.2009.01.022; PII: S0042-6822(09)00047-6; Copyright (c) 2009 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved.; Journal ID: ISSN 0042-6822
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; ALANINES; DNA; MUTATIONS; ONCOGENIC VIRUSES; PHENYLALANINE; PROMOTERS; PROTEINS; AMINO ACIDS; AROMATICS; CARBOXYLIC ACIDS; MICROORGANISMS; NUCLEIC ACIDS; ORGANIC ACIDS; ORGANIC COMPOUNDS; PARASITES; VIRUSES

Citation Formats

Chen, L -W, Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, CT 06520, Raghavan, Vineetha, Chang, Pey-Jium, Shedd, Duane, Heston, Lee, Delecluse, Henri-Jacques, Miller, George, Department of Pediatrics, Yale University School of Medicine, Room 420 LSOG, 333 Cedar St., New Haven, CT 06520, and Department of Epidemiology and Public Health, Yale University School of Medicine, New Haven, CT 06520. Two phenylalanines in the C-terminus of Epstein-Barr virus Rta protein reciprocally modulate its DNA binding and transactivation function. United States: N. p., 2009. Web. doi:10.1016/j.virol.2009.01.022.
Chen, L -W, Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, CT 06520, Raghavan, Vineetha, Chang, Pey-Jium, Shedd, Duane, Heston, Lee, Delecluse, Henri-Jacques, Miller, George, Department of Pediatrics, Yale University School of Medicine, Room 420 LSOG, 333 Cedar St., New Haven, CT 06520, & Department of Epidemiology and Public Health, Yale University School of Medicine, New Haven, CT 06520. Two phenylalanines in the C-terminus of Epstein-Barr virus Rta protein reciprocally modulate its DNA binding and transactivation function. United States. https://doi.org/10.1016/j.virol.2009.01.022
Chen, L -W, Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, CT 06520, Raghavan, Vineetha, Chang, Pey-Jium, Shedd, Duane, Heston, Lee, Delecluse, Henri-Jacques, Miller, George, Department of Pediatrics, Yale University School of Medicine, Room 420 LSOG, 333 Cedar St., New Haven, CT 06520, and Department of Epidemiology and Public Health, Yale University School of Medicine, New Haven, CT 06520. 2009. "Two phenylalanines in the C-terminus of Epstein-Barr virus Rta protein reciprocally modulate its DNA binding and transactivation function". United States. https://doi.org/10.1016/j.virol.2009.01.022.
@article{osti_21357498,
title = {Two phenylalanines in the C-terminus of Epstein-Barr virus Rta protein reciprocally modulate its DNA binding and transactivation function},
author = {Chen, L -W and Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, CT 06520 and Raghavan, Vineetha and Chang, Pey-Jium and Shedd, Duane and Heston, Lee and Delecluse, Henri-Jacques and Miller, George and Department of Pediatrics, Yale University School of Medicine, Room 420 LSOG, 333 Cedar St., New Haven, CT 06520 and Department of Epidemiology and Public Health, Yale University School of Medicine, New Haven, CT 06520},
abstractNote = {The Rta (R transactivator) protein plays an essential role in the Epstein-Barr viral (EBV) lytic cascade. Rta activates viral gene expression by several mechanisms including direct and indirect binding to target viral promoters, synergy with EBV ZEBRA protein, and stimulation of cellular signaling pathways. We previously found that Rta proteins with C-terminal truncations of 30 aa were markedly enhanced in their capacity to bind DNA (Chen, L.W., Chang, P.J., Delecluse, H.J., and Miller, G., (2005). Marked variation in response of consensus binding elements for the Rta protein of Epstein-Barr virus. J. Virol. 79(15), 9635-9650.). Here we show that two phenylalanines (F600 and F605) in the C-terminus of Rta play a crucial role in mediating this DNA binding inhibitory function. Amino acids 555 to 605 of Rta constitute a functional DNA binding inhibitory sequence (DBIS) that markedly decreased DNA binding when transferred to a minimal DNA binding domain of Rta (aa 1-350). Alanine substitution mutants, F600A/F605A, abolished activity of the DBIS. F600 and F605 are located in the transcriptional activation domain of Rta. Alanine substitutions, F600A/F605A, decreased transcriptional activation by Rta protein, whereas aromatic substitutions, such as F600Y/F605Y or F600W/F605W, partially restored transcriptional activation. Full-length Rta protein with F600A/F605A mutations were enhanced in DNA binding compared to wild-type, whereas Rta proteins with F600Y/F605Y or F600W/F605W substitutions were, like wild-type Rta, relatively poor DNA binders. GAL4 (1-147)/Rta (416-605) fusion proteins with F600A/F605A mutations were diminished in transcriptional activation, relative to GAL4/Rta chimeras without such mutations. The results suggest that, in the context of a larger DBIS, F600 and F605 play a role in the reciprocal regulation of DNA binding and transcriptional activation by Rta. Regulation of DNA binding by Rta is likely to be important in controlling its different modes of action.},
doi = {10.1016/j.virol.2009.01.022},
url = {https://www.osti.gov/biblio/21357498}, journal = {Virology},
issn = {0042-6822},
number = 2,
volume = 386,
place = {United States},
year = {Fri Apr 10 00:00:00 EDT 2009},
month = {Fri Apr 10 00:00:00 EDT 2009}
}